Abstract
Fas-associated death domain-containing protein (FADD), a classical apoptotic signaling adaptor, participates in different nonapoptotic processes regulated by its phosphorylation. However, the influence of FADD on metabolism, especially glucose homeostasis, has not been evaluated to date. Here, using both two-dimensional electrophoresis and liquid chromatography linked to tandem mass spectrometry (LC/MS/MS), we found that glycogen synthesis, glycolysis, and gluconeogenesis were dysregulated because of FADD phosphorylation, both in MEFs and liver tissue of the mice bearing phosphorylation-mimicking mutation form of FADD (FADD-D). Further physiological studies showed that FADD-D mice exhibited lower blood glucose, enhanced glucose tolerance, and increased liver glycogen content without alterations in insulin sensitivity. Moreover, investigations on the molecular mechanisms revealed that, under basal conditions, FADD-D mice had elevated phosphorylation of Akt with alterations in its downstream signaling, leading to increased glycogen synthesis and decreased gluconeogenesis. Thus, we uncover a novel role of FADD in the regulation of glucose homeostasis by proteomic discovery and physiological validation.
Highlights
Fas-associated death domain-containing protein (FADD)1, originally identified by the yeast two-hybrid system with the cytoplasmic domain of Fas [1, 2], is considered as an essential mediator of Fas-induced apoptosis [1,2,3]
Using MetaCoreTM pathway analysis tools, we identified a number of differentially expressed proteins involved in glycogen metabolism, glycolysis, and gluconeogenesis, suggesting a novel role for FADD in glucose homeostasis
MetaCoreTM pathway mapping tool revealed that seven proteins among them were involved in glycogen metabolism, glycolysis, and gluconeogenesis processes (Fig. 2 and Table II), which was partially consistent with the above cellular proteomics results
Summary
Stable Cell Line Construction and Cell Culture—WT MEFs and FADD-D MEFs were all cultured in DMEM (Hyclone, Logan, UT) containing 10% FBS (Hyclone) with 50 U/ml penicillin/streptomycin. After filtering through four layers of nylon gauze, the homogenate was centrifuged at 12,000 ϫ g for 10 min, and the supernatant was collected and the protein concentration was determined using the Bradford protein assay kit (Bio-Rad, Hercules, CA). Bioinformatics Analysis—The data set with a list of regulated proteins identified by proteomics was analyzed further by pathway analysis using the network building tool MetaCoreTM version 5.4 (GeneGo, St. Joseph, MI). MetaCoreTM is an integrated software suite for functional analysis of experimental data It is based on a proprietary manually curated database of protein–protein, protein–DNA and protein compound interactions, metabolic and signaling pathways and the effects of bioactive molecules in gene expression. Liver samples were fixed in Carnoy solution (60% ethanol, 30% chloroform, 10% glacial acetic acid) for glycogen detection and stained with Periodic Acid-Schiff (PAS) Kit (SigmaAldrich, St. Louis, MO). Values were considered statistically significant at p Ͻ 0.05
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